Diabetic neuropathy is one of the most important complications afflicting diabetic patients. Since chronic pain caused by diabetic neuropathy often is not adequately relieved by traditional analgesics, it represents an important unmet clinical need. The major objectives of this proposal are to study changes in spinal muscarinic receptors and mechanisms of muscarinic analgesia in diabetic neuropathic pain. Preliminary evidence is presented that muscarinic receptors in the spinal cord are up-regulated in diabetes, which may account for the enhanced muscarinic analgesia in diabetic neuropathic pain. Furthermore, the preliminary study suggests that inhibition of the glutamatergic synaptic input to dorsal horn neurons is an important analgesic mechanism of spinally administered cholinergic agents in diabetic neuropathic pain. The following hypotheses will be tested using animal models of diabetes: 1) Muscarinic receptors in the spinal cord dorsal horn are up-regulated in diabetes; Increased spinal muscarinic M2/M4 receptors play a major role in the enhanced analgesic action of spinally administered cholinergic agents in diabetes; 2) Activation of muscarinic receptors causes a more significant reduction in spinal glutamate release from primary afferent terminals in diabetes; Muscarinic receptor agonists elicit GABA release, which activates presynaptic GABAB receptors to inhibit glutamate release onto spinal lamina II neurons in diabetes; and 3) The inhibitory effects of spinally administered cholinergic agents on spinothalamic tract neurons and nociception are mediated, to a greater extent, by spinal GABAB receptors in diabetes. Quantitative measurements of G protein-coupled receptors, single-unit recordings of spinal dorsal horn neurons, whole-cell voltage-clamp recordings of glutamate- and GABA-mediated postsynaptic currents in spinal cord slices, and behavioral assessment of nociception will be used. These integrated studies are important for our understanding of the mechanisms of altered spinal cord pharmacology in diabetic neuropathic pain. This new information also will provide a rationale for development of improved therapies for patients with diabetic neuropathic pain.